The 300kDa cation-independent mannose 6-phosphate receptor (CI-MPR) and the 46kDa cation-dependent MPR (CD-MPR) interact with a number of different proteins by virtue of their ability to bind mannose 6-phosphate (Man-6-P) residues with high (nM) affinity. The MPRs play a key role in delivering newly synthesized lysosomal enzymes to the lysosome in all higher eukaryotic cells and disruption of this targeting pathway results in the most severe of the human lysosomal storage disorders, mucolipidosis II. In addition to lysosomal enzymes, several growth factors contain Man-6-P and their interaction with the MPRs can result in the growth factor's activation or degradation. The multifunctional CI-MPR also binds several non-Man-6-Pcontaining ligands [insulin-like growth factor-II (IGF-II), urokinase-type plasminogen activator receptor (uPAR), retinoic acid, and plasminogen]. The observation that the CI-MPR regulates normal fetal growth by modulating IGF-II levels and undergoes allelic loss and mutation in a variety of human cancers implicates the CI-MPR as a tumor suppressor gene. Despite the ubiquitous expression of these receptors and the number of different molecules with which they interact, little is known about the molecular basis by which the MPRs recognize their diverse ligands. However, we have made significant progress in understanding the mechanism of carbohydrate recognition by the MPRs: we have provided the first, and to date the only, structural views of the CD-MPR's extracellular domain and the N-terminal three domains of the CI-MPR, both of which house a high affinity Man-6-P binding site and the N-terminal region of the CI-MPR also contains the recently reported uPAR and plasminogen binding site. The five specific aims of this proposal are to determine the structure of the MPRs under conditions which are physiologically relevant using X-ray crystallographic and NMR approaches (aims 1 & 2), to identify residues essential for the interaction of the CI-MPR and plasminogen (aim 3), to evaluate the in vivo role of the newly identified third Man-6-P binding site in domain 5 of the CI-MPR (aim 4), and to determine the crystal structure of the CI-MPR's IGF-II binding site in domain 11 and second high affinity Man-6-P binding site in domain 9 (aim 5). These studies will address our long range goal which is to understand at the structural level the mechanism by which these essential receptors carry out their diverse biological functions.